|Year : 2018 | Volume
| Issue : 8 | Page : 41-45
Effect of inhibition of intermediate-conductance-Ca2+-activated K+ channels on HeLa cell proliferation
Ping Zhan, Ling Liu, Dan Nie, Yaofang Liu, Xiguang Mao
Department of Gynecology, The Affiliated Hospital of Luzhou Medical College, Luzhou, Sichuan, China
|Date of Web Publication||26-Mar-2018|
Department of Gynecology, The Affiliated Hospital of Luzhou Medical College, No. 25 Taiping, Luzhou-646 000, Sichuan
Source of Support: None, Conflict of Interest: None
Purpose: To explore the influence of intermediate-conductance-Ca2+-activated K+ channels. (IKCal) on HeLa cell proliferation.
Materials and Methods: An IKCal blocking agent (clotrimazole (CLT)) and small hairpin ribonucleic acid interference (RNAi) was used to block IKCal in HeLa cells; subsequently, cell growth was observed. Furthermore, the messenger ribonucleic acid (mRNA) expression of IKCal was detected by reverse transcriptase polymerase chain reaction (RT-PCR) after IKCal-blocking.
Results: The obvious morphological changes in HeLa cells were observed 48 h after CLT-blocking. The PCR results indicated that CLT reduced the mRNA expression of IKCal in HeLa cells. HeLa cells were transfected with pGenesil via RNAi; the HeLa cells transfected with pGenesil-IK displayed obvious morphological changes 48 h after transfection. In addition, RT-PCR further demonstrated the reduced mRNA expression of IKCal in the pGenesil group.
Conclusion: CLT and blocking of IKCal gene expression effectively inhibits HeLa cell proliferation; therefore, the use of a blocking agent and RNAi both effectively downregulated the mRNA expression of IKCal, which in turn mediated the proliferation of HeLa cells, producing an antitumor effect.
Keywords: Cell culture, cervical carcinoma, HeLa cells, intermediate-conductance-Ca2+-activated K+ channels
|How to cite this article:|
Zhan P, Liu L, Nie D, Liu Y, Mao X. Effect of inhibition of intermediate-conductance-Ca2+-activated K+ channels on HeLa cell proliferation. J Can Res Ther 2018;14, Suppl S1:41-5
|How to cite this URL:|
Zhan P, Liu L, Nie D, Liu Y, Mao X. Effect of inhibition of intermediate-conductance-Ca2+-activated K+ channels on HeLa cell proliferation. J Can Res Ther [serial online] 2018 [cited 2021 Nov 30];14:41-5. Available from: https://www.cancerjournal.net/text.asp?2018/14/8/41/177212
| > Introduction|| |
Cervical cancer is a common gynecological malignant tumor, whose mechanism remains to be elucidated. Ion channels are pore-forming membrane proteins that allow only ions of a specific size or charge to pass through, via an electrochemical gradient. In addition, ion channels facilitate the establishment of resting membrane and shaping action potentials, and other electrical signals in cells, by gating the flow of ions across the cell membrane and controlling the ion flow across the secretary cells. These ion channels are located in the plasma membrane of almost all cells, and multiple intracellular organelles. Therefore, a majority of current tumor research is focused on ion channels.,, Some studies have identified the widespread distribution of ion channels in a number of organs, and elucidated its important role in maintaining normal physical function and homeostasis. Ion channels include Na + channel, K + channel, Ca 2+ channel and Cl − channel.. Dysfunctions in the K + channels are known to lead to tumor cell proliferation. Activated intermediate-conductance Ca 2+-activated K + channels (IKCal) introduce membranes to a super-polarized state in order to promote extracellular calcium influx, which results in abnormal cell proliferation and a high disease (e.g., tumor) incidence induced by complicated intracellular signaling transduction. Previous studies have discovered high IKCal expressions in endometrial carcinoma, breast cancer, urinary bladder carcinoma, colon cancer, and glioblastoma cells.,,, This study demonstrated the close relationship between the Ca 2+-activated K + channel and cervical cancer., This study utilized the K +-channel blocker clotrimazole (CLT) and small hairpin ribonucleic acid (RNA) to explore the role played by (and the effect of) IKCal in the proliferation of HeLa cells.
| > Materials and Methods|| |
Human cervical cancer cell lines (HeLa) were purchased from Medical University. CLT, Roswell Park Memorial Institute (RPMI) 1640 medium; the restriction enzymes SacI, Eco31I, and T4 DNA ligase; agarose gel kits; the L2000 marker; and the Endo-Free Plasmid Mini Kit I were obtained from Shanghai Sangon Biological Engineering Co., Ltd. (Shanghai, China), and prepared as per the manufacturer protocols. The primers and probes were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. (Shanghai, China). The inverted phase contrast microscope used in this study was obtained from Leica Science Lab Inc. (Berlin, Germany). The JEM1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan) was used in this study. The KCal interference expression vector used in this study was successfully identified by our experimental group in a previous study.
The frozen cells were thawed in a water bath (37–40°C) for 1 min; the thawed cells were cultured in RPMI 1640 medium in a humidified atmosphere with 5% CO2, at 37°C. The culture medium was replaced when the cells adhered to the culture bottle; the experiments were performed until (up to) a maximum of 80% of the cells were adhered.
CLT was dissolved in absolute ethyl alcohol and RPMI 1640 medium containing 10% calf serum, and subsequently diluted to final concentrations of 5, 10, 20, and 40 μmol/L. At 80% confluence, 1 × 103 cells/mL were seeded into 96-well plates, which were cultured for an additional 24 h. Subsequently, the culture medium was replaced with fresh medium, and supplemented with 180 μL CLT at different concentrations (experimental groups) or absolute ethyl alcohol (without CLT; control group); each group was assigned six wells in the plate. The cells were then cultured for 24, 48, and 72 h. The cells were subjected to reverse transcriptase polymerase chain reaction (RT-PCR) in order to detect the messenger RNA (mRNA) expression of IKCal. At a confluence of 80%, 2 × 105 cells/mL were seeded into cultured bottles with RPMI-1640 complete culture medium, and cultured continuously for 24 h; subsequently, these cells were cultured in medium supplemented with 20 μmol/L CLT (experimental group) or pure medium (without CLT; control) for 48 h. The bottles were washed with phosphate buffered saline (PBS), the suspended cells were removed, and the cell morphology observed under an inverted phase contrast microscope. The collected cells were centrifuged and processed (washing, fixation, and embedding) in order to obtain the samples. All sections were observed under a transmission electron microscope (×10,000), and the magnified images were photographed.
RNA interference (RNAi)
At a confluence level of 80%, HeLa cells were transfected with the recombinant plasmids, pGenesil-1.1-IKCal shRNA plasmid (interference sequence), and pGenesil-1.1-HK (negative control sequence) using Lipofectamine JM2000, according to the manufacturer's protocol. The transfected cells were divided into three groups: The pGenesil-IKCal shRNA group, pGenesil-HK group, and the control group. The mRNA expression of IKCal was detected in cells cultured for 24, 48, and 72 h by RT-PCR. The reporter gene expression was detected using green fluorescent protein by fluorescent microscopy, in order to determine the effectiveness of transfection after 48 h; in addition, the cellular morphology was analyzed using an inverted phase contrast microscope.
Total RNA was extracted from the cells of each group using the TRIzol reagent; and the cDNA was synthesized using the RevertAid ™ First Srand cDNA Synthesis Kit (Life Technologies, Carlsbad, CA) as per the manufacturer protocols. The following primers were used for the amplification of IKCal and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): IKCal (856 bp): forward, 5'-GTG CGT GCA GGA TTT AGG G-3' and reverse, 5'-TGC TAA GCA GCT CAG TCA GGG-3'; GAPDH (263 bp): forward, 5'-ATG CTG GCG CTG AGT ACG TC-3' and reverse, 5'-GGT CAT GAG TCC TTC CAC GAT A-3'. PCR was performed under the following reaction conditions: An initial melting step at 95°C for 4 min, 30 cycles of denaturation at 95°C for 45 s, annealing at 57°C for 45 s, and extension at 72°C for 1 min, followed by a final extension step at 72°C for 10 min. The amplified transcripts were subsequently analyzed by 2% agarose gel electrophoresis; the integrated optical density was presented as the ratio of IKCal and GAPDH.
All data was subjected to a statistical analysis using the SPSS v. 13.0 software platform (SPSS Inc, Chicago, IL). The measured data was presented as mean ± standard deviation (SD). The obtained data was compared between groups by Student's t-test, q-test, and one-way analysis of variance (ANOVA). A P value < 0.05 denoted a significant statistical difference.
| > Results|| |
Morphological changes in HeLa cells after CLT treatment
The HeLa cells in the control group were in the shape of a polygon; the cells showed even morphological characteristics with a clear cytoplasm and a nucleus at the center of the cells [Figure 1]a. We observed an abundance of microvilli on the cell surface, in addition to integrated cell membranes and nuclear envelope, organelles, and mitochondria by transmission electron microscopy. In addition, intercellular space, and an abundance of rough endoplasmic reticulum and ribosomes (in the cytoplasm) and euchromatin (in the nucleus) was observed. We observed that the heterochromatin was mottled [Figure 1]b. The size of the cells in the treatment group was smaller and uneven; moreover, the morphology of the treated cells was different, the cytoplasm was unclear, and the size of the nucleus reduced along the side of the cells [Figure 1]c. We observed an obvious reduction in the microvilli of the cell surface, and the nonintegration of the cell membrane and nuclear envelope by transmission electron microscopy. Furthermore, we observed the destruction of the mitochondrion, and a disproportionate ratio of the plasma and nucleus. We also observed an obvious decrease in euchromatin and increase in heterochromatin content (the latter was located on the inside of the nuclear envelope). Additionally, we observed that the intercellular space was enlarged [Figure 1]d.
|Figure 1: Morphological changes of HeLa cells after treatment with clotrimazole. (a) Without treatment (48 h, ×200), (b) without treatment (JEM1400, ×10,000), (c) Treated with 20 μmol/L (JEM1400, ×10,000), and (d) Treated with 20 μmol/L (JEM1400, ×10,000)|
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Morphological changes after transfection
The expression of green fluorescent protein in the transfection and negative control groups was observed under a fluorescent microscope (×200); 10 fields were analyzed for each group in order to determine the number of fluorescent cells, providing a transfection efficiency of 50–60%. However, the group without the plasmid did not express the green fluorescent protein [Figure 2].
|Figure 2: Fluorescence of HeLa cell after transfection under fluorescence microscope (× 200). (a) Transfection group: Fluorescence of HeLa cell after transfection with pGenesil-IK, (b) Negative control group: Fluorescence of HeLa cell after transfection with pGenesil-HK, and (c) Blank control group: Fluorescence of HeLa cell without transfection|
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HeLa cells were cultured in the transfection group after 48 h; transfection resulted in a reduction in the cell size, and roughening of the membrane surface. Furthermore, the treated cells showed shrunken nuclei and clumped chromatin. In addition, the plasma contained a large number of particles and cell fragments. We observed a decrease in the number of adherent cells, with a majority of the cells being suspended in the culture medium. The negative control group showed good HeLa cell growth and a small number of suspended cells. The HeLa cells in the blank control group showed vigorous growth and were shaped as irregular polygons; moreover, these cells displayed a clear cytoplasm, oval and centered nuclei, and similar sizes and morphologies of cells before and after transfection [Figure 3].
|Figure 3: Morphology of HeLa cell after transfection with plasmids under light microscope (×200). (a) Transfection group: HeLa cell after transfection with pGenesil-IK, (b) Negative control group: HeLa cell after transfection with pGenesil-HK, and (c) Blank control group: HeLa cell without transfection|
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We observed an obvious reduction in the relative mRNA expression of IKCal with the increase in CLT concentration and longer reaction times in all experimental groups; the difference was observed to be significantly different at each time point compared to the control group (P < 0.05). Moreover, it showed that the mRNA expression of IKCal was compared with different concentrations and time points, and there was a significant difference between experimental group and control group (P < 0.05, [Table 1] and [Figure 4]).
|Figure 4: Representative RT-PCR result of IKCal in HeLa cell induced by different dose of CLT. M = Marker, 1 = Control group, 2 = 5 μmol/L group, 3 = 10 μmol/L group, 4 = 20 μmol/L, 5 = 40μmol/L, RT-PCR = reverse transcriptase polymerase chain reaction, CLT = clotrimazole, IKCal = intermediate-conductance-Ca2+-activated K+ channels|
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The increase in CLT concentration and extension in reaction time resulted in an obvious reduction in the relative IKCal mRNA expression in all experimental groups; in addition, the relative IKCal mRNA expression was significantly different at any time point in the experimental groups compared to the negative and control groups (P < 0.05). However, no obvious change in IKCal mRNA expression was observed in the negative control and blank control groups, and the difference was not significant at any time point (P > 0.05) [Figure 5] and [Table 2].
|Figure 5: Representative RT-PCR result of IKCal in HeLa cell induced with transfection. M = Maker, 1 = pGenesil-IK1, 2 = pGenesil-IK2, 3 = pGenesil-HK, 4 = HeLa|
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|Table 2: Expression of IKCal mRNA in HeLa cell induced by transfected with plasmids|
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| > Discussion|| |
K + channels are the most complicated ion channels in cells; there are several types of K + channels, including the Ca 2+-activated, Na +-activated, and mechanically activated K + channels. The IKCal is a key component of the potassium channel family, which regulates the membrane potential and the concentration of intracellular Ca 2+, in addition to mediating multiple cellular biological activities, such as cytokine secretion, electrolyte transportation, permeation pressure maintenance, cell migration, and proliferation.,,, Recent studies have demonstrated aberrant (high) expressions of KCal genes in multiple tumor tissues and cells, which promoted tumor cell proliferation.,,
CLT, a KCal blocker, has seen widespread use. Parihar et al., observed that, while the KCal activator 1-EBIO promoted LNCaP and PC-3 cell proliferation, CLT inhibited the 1-EBIO-induced increase in proliferation of LNCaP and PC-3 cells. Furthermore, Jäger et al., observed that the KCal blocker inhibited BxPC-3 and MiaPaCa-2 cell proliferation; the blockage of KCal converted cells to depolarized states, with the decrease in intracellular calcium concentration resulting in the cells being arrested in the G0/G1 phase. This study clearly showed the reduced size, discontinuous membranes, opaque cytoplasm, and endonuclear chromatin accumulation in HeLa cells treated with CLT. The increase in CLT concentration and extension in reaction time led to an increase in inhibition of HeLa cell growth, and reduced IKCal mRNA expression. Interestingly, the morphological changes observed in the cells after RNAi (to silence the IKCal gene) of HeLa cells were similar to those observed upon treatment with CLT, which indicated that cell proliferation was affected by the blocking of IKCal by CLT. Zhang et al., demonstrated that the KCa3.1 specific antagonist TRAM-34 suppressed the proliferation of HEC-1-A and Ishikawa cells in a time- and dose-dependent manner. In addition, RNAi significantly inhibited the protein expressions of KCa3.1 channels, and the proliferation of HEC-1-A and Ishikawa cells. Haren et al., analyzed breast cancer tissues and cells by immunohistochemistry, quantitative real-time RT-PCR, western blot assay, and the whole cell patch clamp technique, and observed the high expression of IKCal in these cells; in addition, higher expressions of the hKCa3.1 mRNA and proteins were observed in grade III tumors, compared to both grade I and II tumors. A clinical pathological evaluation indicated a significant correlation between hKCa3.1-expression and the tumor grade. In our previous studies, we have observed higher mRNA expression of IKCal in cervical cancer tissues compared to normal tissues;, the results of this study further indicated an obvious decrease in the IKCal mRNA expression in the transfection group (in which the IKCal gene was blocked or silenced), compared to the negative and control groups. This result indicated that the cell potential and energy transfer was affected after blockage of IKCal and silencing of the IKCal gene; in addition, we believe that the permeation pressure was also altered to influence the growth of cells so as to reduce cell count, or even induce cell necrosis (resulting from low nutrition). These results were similar to those observed by De Marchi et al., who reported that a moderate increase in mitochondrial matrix Ca 2+ resulted in the opening of the mtKCa3.1 channel.
In summary, the inhibition of KCal activity could regulate the intracellular and extracellular Ca 2+ concentrations, suppress or activate some signal pathway, destroy cellular energy metabolism, and control the expression of cell cycle-related proteins, cumulatively affecting the cell proliferation. On the basis of the mechanisms supported by IKCal, and the regulation of cell growth by IKCal, research and development into drugs that specifically inhibit KCal expression in cervical cancer would help improve the diagnosis, therapy, and prognosis of cervical cancer.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Guéguinou M, Chantôme A, Fromont G, Bougnoux P, Vandier C, Potier-Cartereau M. KCa and Ca 2+
channels: The complex thought. Biochim Biophys Acta 2014;1843:2322-33.
Weisbrod D, Peretz A, Ziskind A, Menaker N, Oz S, Barad L, et al
., SK4 Ca 2+
activated K +
channel is a critical player in cardiac pacemaker derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2013;110:E1685-94.
Hayashi M, Wang J, Hede SE, Novak I. An intermediate-conductance Ca 2+
-activated K+channel is important for secretion in pancreatic duct cells. Am J Physiol Cell Physiol 2012;303:C151-9.
Wang ZH, Feng YJ, Su M, Yi XF. Intermediate-conductance-Ca 2+
-activated K +
channels are overexpressed in endometrial cancer and involved in regulating proliferation of endometrial cancer cells. Zhonghua Fu Chan Ke Za Zhi 2007;42:111-5.
Haren N, Khorsi H, Faouzi M, Ahidouch A, Sevestre H, Ouadid-Ahidouch H. Intermediate conductance Ca 2+
activated K +
channels are expressed and functional in breast adenocarcinomas: Correlation with tumour grade and metastasis status. Histol Histopathol 2010;25:1247-55.
Nielsen JS, Rode F, Rahbek M, Andersson KE, Rønn LC, Bouchelouche K, et al
., Effect of the SK/IK channel modulator 4, 5-dichloro-1, 3-diethyl-1, 3-dihydro-benzoimidazol-2-one (NS4591) on contractile force in rat, pig and human detrusor smooth muscle. BJU Int 2011;108:771-7.
Maroto R, Kurosky A, Hamill OP. Mechanosensitive Ca (2+) permeant cation channels in human prostate tumor cells. Channels (Austin) 2012;6:290-307.
Judge SI, Smith PJ, Stewart PE, Bever CT Jr. Potassium channel blockers and openers as CNS neurologic therapeutic agents. Recent Pat CNS Drug Discov 2007;2:200-28.
Shen MR, Chou CY, Ellory JC. Swelling-activated taurine and K +
transport in human cervical cancer cells: Association with cell cycle progression. Pflugers Arch 2001;441:787-95.
Adelman JP, Maylie J, Sah P. Small-conductance Ca 2+
-activated K +
channels: Form and function. Annu Rev Physiol 2012;74:245-69.
N'Gouemo P. Targeting BK (big potassium) channels in epilepsy. Expert Opin Ther Targets 2011;15:1283-95.
Berkefeld H, Fakler B, Schulte U. Ca 2+
-activated K +
channels: From protein complexes to function. Physiol Rev 2010;90:1437-59.
Barfod ET, Moore AL, Roe MW, Lidofsky SD. Ca2+-activated IK1 channels associate with lipid rafts upon cell swelling and mediate volume recovery. J Biol Chem 2007;282:8984-93.
Jäger H, Dreker T, Buck A, Gress T, Grissmer S. Blockage of intermediate-conductance Ca 2+
-activated K +
channels inhibit human pancreatic cancer cell growth in vitro
. Mol Pharmacol 2004;65:630-8.
Lallet-Daher H, Roudbaraki M, Bavencoffe A, Mariot P, Gackière F, Bidaux G, et al
., Intermediate-conductance Ca 2+
-activated K +
channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry. Oncogene 2009;28:1792-806.
Roy JW, Cowley EA, Blay J, Linsdell P. The intermediate conductance Ca 2+
-activated K +
channel inhibitor TRAM-34 stimulates proliferation of breast cancer cells via activation of oestrogen receptors. Br J Pharmacol 2010;159:650-8.
Kast RE. Profound blockage of CXCR4 signaling at multiple points using the synergy between plerixafor, mirtazapine, and clotrimazole as a new glioblastoma treatment adjunct. Turk Neurosurg 2010;20:425-9.
Parihar S, Coghlan MJ, Gopaiakrishnan M, Shieh CC. Efiects of intermediate-conductance-Ca 2+
-activated K +
channel modulators on haman prostate cancer cell proliferation. Eur J Pharmacol 2003;471:157-64.
Zhang YL, Zhao MZ, Lu X, Huang Y, Yi XF, Yu YH, et al
., Effect of expression of intermediate-conductance Ca 2+
-activated K +
channels on proliferation, cell cycle, and apoptosis of human endometrial carcinoma. Tumor 2009;29:323-8.
De Marchi U, Sassi N, Fioretti B, Catacuzzeno L, Cereghetti GM, Szabò I, et al
., Intermediate conductance Ca 2+
-activated potassium hannel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells. Cell Calcium 2009;45:509-16.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]